The present invention relates first, to the identification of a novel human retrovirus and the novel nucleotide sequences encoding a retroviral long terminal repeat and reverse transcriptase nucleotides associated with the existence of primary biliary cirrhosis (PBC), and other immune disorders such as Sjxc3x6gren""s syndrome, scleroderma, systemic lupus erythematosus (SLE), autoimmune thyroiditis and various other connective tissue disorders, in addition to lymphoma and breast cancer. The present invention further relates to methods for using the PBC retroviral nucleotides for the detection of PBC, Sjxc3x6gren""s syndrome, scleroderma, SLE, autoimmune thyroiditis and various other connective tissue disorders in patient samples. The present invention also relates to methods for using and targeting the PBC retroviral long terminal repeat and reverse transcriptase nucleotides in gene therapy protocols for the treatment of PBC, Sjxc3x6gren""s syndrome, scleroderma, SLE, autoimmune thyroiditis and various other connective tissue disorders in patients in need of such treatment. The present invention also relates to methods of treating or inhibiting PBC retroviral infection with antiviral agents, such as cytokines, inhibitors of reverse transcriptase, inhibitors of viral capping, and inhibitors of viral protease. The present invention further relates to diagnostic protocols and kits for the detection of PBC, Sjxc3x6gren""s syndrome, scleroderma, SLE, autoimmune thyroiditis and various other connective tissue disorders in tissue samples.
Primary Biliary Cirrhosis and Autoimmunity
Primary biliary cirrhosis (PBC) is a progressive pluriglandular disease affecting the liver, pancreas, salivary and lachrymal glands (Neuberger, 1997, Lancet 850:875-79; Epstein et al., 1980, Lancet 1:1166-68). The hepatic disease is characterized by a florid bile duct lesion with lymphocytic infiltration and granulomatous destruction of 30 to 80 xcexcm sized interlobular bile ducts (Rubin et al., 1965, Am. J. Pathol. 46:387-407). There is no curative therapy, apart from liver transplantation, and patients usually develop cirrhosis (Neuberger et al., 1997, Lancet 250:875-879). It is estimated to account for approximately 2% of patients dying from cirrhosis in Europe and 10% of patients that requiring orthotopic liver transplantation in North America (Neuberger et al., 1997, Lancet 250:875-879).
PBC is considered an archetypal autoimmune disease because patients have anti-mitochondrial antibodies (AMA) and can also present with other autoimmune disorders such as Sjxc3x6gren""s syndrome, scleroderma, systemic lupus erythematosus (SLE), autoimmune thyroiditis, and various other connective tissue disorders (Neuberger et al., 1997, Lancet 250:875-879). The clinical overlap with Sjxc3x6gren""s syndrome is particularly marked, as tests for xerophthalmia and xerostomia are positive in 70% to 100% of PBC patients (Alarcon-Segovia et al., 1973, Ann. Intern. Med. 79:31). In addition, patients with PBC have an increased incidence of urinary tract infection (Burroughs et al., 1984, Gut 25:133-7) and a 4 fold increased risk of breast cancer (Wolke et al., 1984, Am. J. Med. 76:1075).
The autoimmune phenomena associated with PBC have been well characterized. Over 95% of PBC patients have antibodies that bind and inhibit the dihydrolipoamide acetyltransferase enzymatic function of the E2 subunit of the pyruvate dehydrogenase complex (PDC) (Gershwin et al., 1992, Molecular biology of the 2-oxo-acid dehydrogenase complexes and anti-microbial antibodies. Philadelphia W. B. Saunders) These AMA have a higher affinity to the dehydrogenase E2 enzymes of mammals as compared to invertebrates and react to the E2 sub-units of other highly conserved inner membrane mitochondrial proteins of the oxoglutarate dehydrogenase complex, and branched chain 2-oxoacid dehydrogenase complex, and also the E1xcex1 and E1xcex2 sub-units of PDC. For patients with liver disease, reactivity to the E2 mitochondrial enzymes is specific to PBC but these AMA have been observed in individuals with Sjxc3x6gren""s syndrome and SLE as well (Van-de-Water et al., 1989, New Eng. J. Med. 320: 1377-80). The reason why PBC patients have an antigen driven immune response to human PDC-E2 may be partially explained by the findings of immunohistochemical studies. Using monoclonal and combinatorial AMA, PDC-E2 or antigens resembling PDC-E2 have been observed on the surface of cultured PBC biliary epithelium cells (Joplin et al., 1992, Lancet 339: 93-94), biliary epithelium and lymph node macrophages in PBC patient""s tissues (Joplin et al., 1991, Hepatology 14: 442-447, Van-de-Water et al., 1993, J. Clin. Invest. 91: 2654-64), and salivary glands of patients with PBC and Sjxc3x6grens syndrome (Tsuneyama et al., 1994, Hepatology 20: 893-898). In essence, the tissues affected by the pluriglandular disease process are the same as those with the abnormal distribution of PDC-E2 antigens on epithelial cell surface.
The disease has been observed in all races and predominantly affects women (Neuberger, 1997, Lancet 350: 875-879). To date, non-HLA genetic factors predisposing to PBC have not been identified but a positive family history provides the greatest risk of developing disease (Sherlock et al., 1993, Primary biliary cirrhosis: definition and epidemiological features. Kluwer Academic Publishers, Doredrecht/Boston/London, pp. 341-49). There are well documented cases of clustering in families and one report documented a 2.4% familial prevalence (Sherlock et al., 1993, Primary biliary cirrhosis: definition and epidemiological features. Kluwer Academic Publishers, Doredrecht/Boston/London. 341-49 pp). No HLA class I alleles are associated with PBC but other immunogenetic factors appear to play an important role.
There are limited data to suggest an infectious etiology of PBC. The spread of disease has been documented in unrelated care providers and has also been associated with a particular water supply in Sheffield, England which was reconfirmed with a follow-up study 10 year later (Sherlock et al., 1993, Primary biliary cirrhosis: definition and epidemiological features. Kluwer Academic Publishers, Doredrecht/Boston/London. pp. 341-49, Trigger 1980, Br. Med. J. 281: 772-5). Although the data are subject to debate, the specific anti-mitochondrial antibodies associated with PBC have been detected in related and non-related family members (Sherlock et al., 1993, Primary biliary cirrhosis: definition and epidemiological features. Kluwer Academic Publishers, Doredrecht/Boston/London, 341-49). Further evidence for an infectious etiology of PBC is suggested by the observation of recurrent disease in the hepatic allograft of approximately 15% of PBC patients undergoing orthotopic liver transplantation. This evidence includes the observation of granulomatous destruction of bile ducts after liver transplantation, the continued presence of serum AMA in the majority of PBC patients (Trigger, 1980, Br. Med. J. 281: 772-5), and immunohistochemical evidence of PDH-E2 on biliary epithelial cell surface in the allograft (Neuberger et al., 1982, N. Eng. J. Med. 306: 1-4).
The epidemiology of PBC does not suggest a simple infectious disease pattern. If an infectious etiology is entertained, it is probable that infection only causes PBC in predisposed individuals due to the modulating effects of genetic, hormonal, and environmental factors. This may partially explain why PBC patients develop AMA and liver disease while other family members develop merely develop serum AMA reactivity without PBC. Many investigators have postulated that either mycobacteria, or enterobacterial R-forms are etiologically related to PBC as they have the highly conserved dehydrogenase enzymes which possibly induce autoimmunity by the mechanism of microbial molecular mimicry with host proteins (O""Donohue et al., 1994, J. Hepatol. 21: 887-889; Stemerowicz et al., 1988, Lancet 1: 1166-1170). Others have made the claim that recurrent bacterial urinary tract infections may be the source of such antigen exposure (Burroughs et al., 1984, Gut 25: 133-7), but this hypothesis provides no explanation for limited disease restricted to biliary epithelium with an immune response to a ubiquitous autoantigen. Thus, the involvement of an infectious agent with PBC still remains to be characterized.
The present invention relates, first, to the discovery, identification, and characterization of novel nucleic acid molecules, that are associated with PBC. The novel nucleotide sequences of the present invention are retroviral in origin and are indicative of a PBC retrovirus which bears a strong correlation with PBC. The present invention is based, in part, on the Applicants"" data which is the first evidence to suggest that PBC patient""s tissue may harbor a transmissible agent. The association of a retroviral infectious agent with PBC was first demonstrated by Applicants in vitro by co-culture of periportal lymph nodes derived from patients at time of transplantation and healthy biliary epithelium cells. The Applicants"" discoveries as described herein, report the characterization of PBC-associated infectious agent as retroviral as demonstrated by electron microscopy and immunoblot reactivity. In addition, Applicants have characterized novel nucleotide sequences which are associated with the PBC-associated retrovirus.
The present invention encompasses nucleic acid molecules which comprise the following nucleotide sequences: (a) nucleotide sequences comprising the PBC retroviral sequences disclosed herein; and (b) nucleotide sequences that encompass portions or fragments of the PBC retroviral nucleotides which can be utilized as probes or primers in the methods of the invention for identifying and diagnosing individuals at a risk for, or exhibiting PBC, Sjxc3x6gren""s syndrome, scleroderma, SLE, autoimmune thyroiditis, various other connective tissue disorders and lymphoma.
The invention also encompasses the expression products of the nucleic acids molecules listed above; i.e., proteins and/or polypeptides that are encoded by the above PBC retroviral nucleic acid molecules, or by degenerative, e.g., allelic variants thereof.
The compositions of the present invention further encompass antagonists of the PBC retroviral gene products, including small molecules, large molecules, and antibodies, as well as nucleotide sequences that can be used to inhibit PBC retroviral gene expression, e.g., antisense, ribozyme molecules, and gene or regulatory sequence replacement constructs.
The present invention relates to therapeutic methods and compositions for treatment and prevention of diseases and disorders associated with the presence of the PBC retroviral nucleotides, including but not limited to, PBC, Sjxc3x6gren""s syndrome, scleroderma, SLE, autoimmune thyroiditis and various other connective tissue disorders. The therapeutic methods and compositions of the present invention are designed to target the PBC retroviral nucleotides, such as antisense molecules and ribozymes. The therapeutic methods and compositions of the present invention are also designed to target PBC retroviral gene products, including small molecules, large molecules, and antibodies. The present invention further relates to the vaccine formulations based on isolated PBC associated virus particles in an attenuated form and/or PBC retroviral gene products for the treatment and/or prevention of disorders associated with the presence of PBC retroviral nucleotides.
In addition, the present invention is directed to methods that utilize the nucleotide sequences of the present invention for the diagnostic evaluation, genetic testing and prognosis of PBC retroviral infection and/or associated disorders including, (but not limited to PBC, Sjxc3x6gren""s syndrome, scleroderma, SLE, autoimmune thyroiditis and various other connective tissue disorders. For example, in one embodiment, the invention relates to methods of diagnosing PBC retroviral infection and/or associated disorders, wherein such methods comprise measuring PBC retroviral gene expression in a patient sample suspected of exhibiting such a disorder. In one embodiment, nucleic acid molecules of the present invention can be used as primers for diagnostic PCR analysis for the identification of PBC retroviral nucleotides which correlate with the presence of a PBC retrovirus and/or associated disorders PBC, Sjxc3x6gren""s syndrome, scleroderma, SLE, autoimmune thyroiditis, various other connective tissue disorders and lymphoma. In yet another embodiment, the nucleic acid molecules of the present invention may be used to detect breast cancer in a subject shown also to be infected with PBC retrovirus in tissue samples other than breast tissue e.g., liver tissue or serum. In another embodiment, antibodies or serologic assays may be used to detect breast cancer in a subject shown to be also infected with PBC in tissue samples other than breast tissue, e.g., liver tissue or serum samples. In yet another embodiment, nucleic acid molecules of the present invention can be used as primers for therapeutic PCR analysis in order to monitor the presence of a PBC retrovirus in order to determine the effectiveness of a therapeutic protocol.
In yet another embodiment, the present invention relates to diagnostic evaluation and prognosis methods which are based on immuno reactivity of a patient sample to PBC-specific antibodies. In this embodiment, isolated serum from a PBC-positive patient, monoclonal antibodies or polyclonal antibodies having specificity for PBC-associated virus may be used to detect PBC-associated retrovirus in a patient sample as a method of diagnosis or as a method of determining the effectiveness of a therapeutic protocol.
In yet another embodiment, the present invention relates to methods of treating individual, infected with PBC retrovirus. In particular, the present invention relates to combinations of antiviral and immunomodulation therapy to control viral replication and disease symptoms in individuals infected with PBC retrovirus the present invention further relates to vaccines and other prophylectic treatments to prevent disease in genetically susceptible individuals.
3.1 Definitions
xe2x80x9cComplementxe2x80x9d or xe2x80x9ctag complementxe2x80x9d as used herein in reference to oligonucleotide tags refers to an oligonucleotide to which a oligonucleotide tag specifically hybridizes to form a perfectly matched duplex or triplex. In embodiments where specific hybridization results in a triplex, the oligonucleotide tag may be selected to be either double stranded or single stranded. Thus, where triplexes are formed, the term xe2x80x9ccomplementxe2x80x9d is meant to encompass either a double stranded complement of a single stranded oligonucleotide tag or a single stranded complement of a double stranded oligonucleotide tag.
The term xe2x80x9coligonucleotidexe2x80x9d as used herein includes linear oligomers of natural or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, a-anomeric forms thereof, peptide nucleic acids (PNAs), and the like, that are capable of specifically binding to a target polynucleotide. The specific binding is determined by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like. Usually monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides that range in size from a few monomeric units, e.g. 3-4, to several tens of monomeric units. Whenever an oligonucleotide is represented by a sequence of letters, such as xe2x80x9cATGCCTG,xe2x80x9d it will be understood that the nucleotides are in 5xe2x80x2 greater than 3xe2x80x2 order from left to right and that xe2x80x9cAxe2x80x9d denotes deoxyadenosine, xe2x80x9cCxe2x80x9d denotes deoxycytidine, xe2x80x9cGxe2x80x9d denotes deoxyguanosine, and xe2x80x9cTxe2x80x9d denotes thymidine, unless otherwise noted. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoranilidate, phosphoramidate, and the like. It is clear to those skilled in the art when oligonucleotides having natural or non-natural nucleotides may be employed, e.g. where processing by enzymes is called for, usually oligonucleotides consisting of natural nucleotides are required. xe2x80x9cPerfectly matchedxe2x80x9d in reference to a duplex means that the poly- or oligonucleotide strands making up the duplex form a double stranded structure with one other such that every nucleotide in each strand undergoes Watson-Crick basepairing with a nucleotide in the other strand. The term also comprehends the pairing of nucleoside analogs, such as deoxyinosine, nucleosides with 2-aminopurine bases, and the like, that may be employed. In reference to a triplex, the term means that the triplex consists of a perfectly matched duplex and a third strand in which every nucleotide undergoes Hoogsteen or reverse Hoogsteen association with a basepair of the perfectly matched duplex. Conversely, a xe2x80x9cmismatchxe2x80x9d in a duplex between a tag and an oligonucleotide means that a pair or triplet of nucleotides in the duplex or triplex fails to undergo Watson-Crick and/or Hoogsteen and/or reverse Hoogsteen bonding. It also includes known types of modifications, for example, labels which are known in the art, methylation, xe2x80x9ccapsxe2x80x9d, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example proteins (including for e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide.
As used herein, xe2x80x9cnucleosidexe2x80x9d includes the natural nucleosides, including 2xe2x80x2-deoxy and 2xe2x80x2-hydroxyl forms, e.g. as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). xe2x80x9cAnalogsxe2x80x9d in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g. described by Scheit, Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990), or the like, with the only proviso that they are capable of hybridization. Such analogs include synthetic nucleosides designed to enhance binding properties, reduce degeneracy, increase or decrease specificity, and the like.
As used herein, a polynucleotide xe2x80x9cderived fromxe2x80x9d a designated sequence refers to a subset of the designated sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides. xe2x80x9cCorrespondingxe2x80x9d means homologous to or complementary to the designated sequence. Preferably, the sequence of the region from which the polynucleotide is derived is homologous to or complementary to a sequence which is unique to an PBC associated viral genome. More preferably, the derived sequence is homologous or complementary to a sequence that is unique to all or to a majority of PBC associated viral isolates. Whether or not a sequence is unique to the a PBC associated viral genome can be determined by techniques known to those of skill in the art. For example, the sequence can be compared to sequences in databanks, e.g., Genebank, to determine whether it is present in the uninfected host or other organisms. The sequence can also be compared to the known sequences of other viral agents, including retroviruses. The correspondence or non-correspondence of the derived sequence to other sequences can also be determined by hybridization under the appropriate stringency conditions. Hybridization techniques for determining the complementarity of nucleic acid sequences are known in the art, and are discussed infra. See also, for example, Maniatis et al. (1982). In addition, mismatches of duplex polynucleotides formed by hybridization can be determined by known techniques, including for example, digestion with a nuclease such as S1 that specifically digests single-stranded areas in duplex polynucleotides. Regions from which typical DNA sequences may be xe2x80x9cderivedxe2x80x9d include but are not limited to, for example, regions encoding specific epitopes, as well as non-transcribed and/or non-translated regions.
The derived polynucleotide is not necessarily physically derived from the nucleotide sequence shown, but may be generated in any manner, including for example, chemical synthesis or DNA replication or reverse transcription or transcription. In addition, combinations of regions corresponding to that of the designated sequence may be modified in ways known in the art to be consistent with an intended use.
The term xe2x80x9crecombinant polynucleotidexe2x80x9d as used herein intends a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin. The term further intends that the polynucleotide (1) is not associated with all or a portion of a polynucleotide with which it is associated in nature; (2) is linked to a polynucleotide other than that to which it is linked in nature; or (3) does not occur in nature.
As used herein, the xe2x80x9csense strandxe2x80x9d of a nucleic acid contains the sequence that has sequence homology to that of mRNA. The xe2x80x9canti-sense strandxe2x80x9d contains a sequence which is complementary to that of the xe2x80x9csense strandxe2x80x9d.
The term xe2x80x9cprimerxe2x80x9d as used herein refers to an oligomer which is capable of acting as a point of initiation of synthesis of a polynucleotide strand when placed under appropriate conditions. The primer will be completely or substantially complementary to a region of the polynucleotide strand to be copied. Thus, under conditions conducive to hybridization, the primer will anneal to the complementary region of the analyte strand. Upon addition of suitable reactants, (e.g., a polymerase, nucleotide triphosphates, and the like), the primer is extended by the polymerizing agent to form a copy of the analyte strand. The primer may be single-stranded, or alternatively may be partially or fully double-stranded.
The terms xe2x80x9canalyte polynucleotidexe2x80x9d and xe2x80x9canalyte strandxe2x80x9d refer to a single- or double-stranded nucleic acid molecule which is suspected of containing a target sequence, and which may be present in a biological sample. As used herein, the term xe2x80x9coligomerxe2x80x9d refers to primers and to probes. The term oligomer does not connote the size of the molecule.
As used herein, the term xe2x80x9cprobexe2x80x9d refers to a structure comprised of a polynucleotide which forms a hybrid structure with a target sequence, due to complementarity of at least one sequence in the probe with a sequence in the target region. The polynucleotide regions of probes may be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs. Included within probes are xe2x80x9ccapture probesxe2x80x9d and xe2x80x9clabel probesxe2x80x9d. Preferably the probe does not contain a sequence complementary to sequence(s) used to prime the polymerase chain reaction (PCR).
As used herein, the term xe2x80x9ctarget regionxe2x80x9d refers to a region of the nucleic acid which is to be amplified and/or detected. The term xe2x80x9ctarget sequencexe2x80x9d refers to a sequence with which a probe or primer will form a stable hybrid under desired conditions.
As used herein, the term xe2x80x9cviral RNAxe2x80x9d, which includes PBC associated RNA, refers to RNA from the viral genome, fragments thereof, transcripts thereof, and mutant sequences derived therefrom.
As used herein, a xe2x80x9cbiological samplexe2x80x9d refers to a sample of tissue or fluid isolated from an individual. Thus, xe2x80x9cbiological samplexe2x80x9d, includes but is not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs, and also samples of in vitro cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, putatively virally infected cells, recombinant cells, and cell components).